Focoidan Inhibits Activation Of NLRP3 Inflammasome In Uric Acid-exposed HK-2 Cells And Its Mechanism

Uric acid(UA)is the end product of purine nucleotide metabolism in human body.Reduction of uric acid excretion results in high blood uric acid level or urate crystals deposition,and consequent diseases,such as hyperuricemia and gout.Kidney is the main organ for excretion of uric acid and regulation of blood uric acid.And renal tubular epithelial cells are one of the targets directly affected by uric acid.Excessive blood uric acid can activate pro-inflammatory cytokines and cause inflammatory injury.Fucoidan(FPS)is a marine drug with a variety of biological activities,and has been used in the clinical treatment of nephropathy for nearly 20 years.At present,there have been many reports on the effect of FPS on UA-induced renal injury and cellular inflammatory response,but few studies have explored the inhibitory effect of FPS on UA induced cellular inflammatory response.Based on human renal tubular epithelial cells(HK-2),this study investigated inhibition of FPS on activation of NLRP3 inflammasome and the involved mechanisms,including NF-κB and MAPKs(ERK 1/2,p38 MAPK,JNK)signaling pathways,in UA exposed HK-2 cells.HK-2 cells were exposed to UA and FPS,and then subjected to assess content of NO,activity of iNOS and content of NLRP3 protein.Results revealed that UA increased NO content,iNOS activity and NLRP3 protein expression in a dose-dependent manner,however the negative effects were inhibited by FPS exposure.Inhibitors of NF-κB and MAPKs(ERK 1/2,JNK,p38 MAPKs)signaling pathways were used to explore the mechanism of FPS inhibits UA-induced NLRP3 inflammasome activation.The results showed that NF-κB and MAPKs inhibitors decreased the phosphorylation level of corresponding pathway proteins in normal cells,but did not affect the expression of inflammatory factors.UA causes cellular inflammatory response by activating NF-κB,ERK 1/2,JNK and p38 MAPK signaling pathways and up-regulating the expression of NLRP3,ASC,Caspase-1 and IL-18.FPS had no effect on NF-κB and MAPKs pathways and pro-inflammatory factors in normal cells,but it respressed activation of pathways proteins and NLRP3 inflammasome,and expression of pro-inflammatory factors induced by UA,consequently protected cells from UA induced cytotoxicity.These results indicated that NF-κB and MAPKs signaling pathways were involved in UA-induced inflammatory response in HK-2 cells,and FPS inhibits the activation of NLRP3 inflammasome and down-regulate the expression of pro-inflammatory factors by inhibiting the activation of NF-κB and MAPKs signaling pathways,thus alleviating cellular inflammation.In this study,we also found that UA promote the expression of GSDMD protein in cells,while FPS inhibit the effect through NF-κB and MAPKs signaling pathways,suggesting that FPS may be effective to inhibit UA-induced pyroptosis of HK-2 cells,but it should be testified by further study.In conclusion,FPS effectively inhibit the activation of NF-κB and MAPKs signaling pathways and the expression of GSDMD protein in UA exposed HK-2 cells,as well as the expression of NLRP3,ASC,Caspase-1 and IL-18 inflammatory factors.These results suggested that the mechanism of FPS inhibits UA-induced NLRP3 inflammasome in HK-2cells involves NF-κB and MAPKs signaling pathways.